Abstract: Epigenetic modifications play an important role in gene transcription regulation. One typical example of these epigenetic modifications is DNA methylation. Methylation of DNA is mainly occurred at the 5 position of the cytosine pyrimidine ring in a CpG dinucleotide context, which is catalyzed by DNA methyltransferases. DNA methylation at gene promoter region and transcription start sites regulates gene transcription. Recently, TET family enzymes were shown to oxidize the methylated cytosine in a step towards DNA demethylation. These enzymes specifically convert methylated cytosine (5mC) mainly into hydroxymethylated cytosine (5hmC). Interestingly, unlike DNA demethylation, TET enzymes-dependent oxidation of methylated cytosine is not only associated with transcription activation but also transcription repression. It has been reported that TET1 forms a complex with SIN3A and HDAC1/2, which is involved in transcription repression. To study the molecular mechanism of TET enzyme-dependent DNA demethylation, we examined the associated proteins of TET enzymes using an unbiased protein affinity purification approach, and found OGT as a functional partner of TET2 in mouse ES cells. OGT is the only enzyme that uses UDP-GlcNAc as the donor to catalyze protein O-GlcNAcylation. Our preliminary study shows that OGT interacts with TET2 to form a heterodimer and is targeted to chromatin for histone GlcNAcylation via TET2 in mouse ES cells. Genome- wide profiling of TET2 and OGT suggests that TET2, OGT and OGT-dependent histone GlcNAcylation are associated with active gene transcription in mouse ES cells. Moreover, OGT-dependent histone GlcNAcylation regulates histone H2B ubiquitiation, an active gene transcription mark. Thus, we hypothesize that the OGT/TET2 complex is involved in transcription activation and counteracts TET1-dependent gene silencing. We plan to perform following studies to test our hypothesis. Aim1: To examine the role of the TET2/OGT complex in H2B ubiquitination. Aim2: To investigate the role of the TET2/OGT complex in histone acetylation. Aim3: To study the functional interaction between the TET1 complex and the TET2 complex in transcription regulation. Taken together, the proposed study will comprehensively examine the function of TET enzyme-dependent gene transcription. It might provide novel molecular mechanisms of trans-tail histone modifications that are important for transcription activation.